| Background and aim The main mechanism for inhibition of TSGs in CRC is DNA hypermethylation.5hmC is an intermidate during the progress of demethylation,however,conventional DNA methylation technology cannot distinguish between 5mC and 5hmC.Thus,accurate analysis of global DNA methylation and hydroxymethylation in CRC is benefit for discovery of genes associated with CRC.TET enzyme is an important demethylase discovered recently.It was known that TET1 reactive TSGs by depressing DNA methylation,besides,TET1 transcription was regulated by poly-ADP-ribosylation.However,it is unknown whether mono-ADP-ribosylation of histone,the fundamental of poly-ADP-ribosylation of histone,regulates DNA methylation of TSGs via TET1 in CRC.Methods PART 1: Applying HMST-seq to globally detect patterning of both 5mC and 5hmC synchronously,in 16 pairs of CRC samples and their adjacent non-cancerous normal tissues.Then,performing functional analysis of genes containing differentially hydroxymethylated or methylated regions by WEB-based GEne SeT AnaLysis Toolkit(WebGestalt)to screen genes,which associated with CRC.PART 2: Based on the results of PART 1,we selected TFPI2 as research gene and H3R117 A Lo Vo cells,which was established successfully in previous study,as experimental group,discussing effect and mechanism of mono-ADP-ribosylation of H3R117 on TFPI2 methylation by regulating TET1 in CRC.MeDIP-qPCR and hMeDIP-qPCR were used for testing methylated and hydromethylated level of TFPI2 promoter,respectively;and ChIP assay was used for measuring enrichment of PAR,TET1,H3K4me3 and H3K9me3 on TFPI2 promoter and enrichment of PAR,PARP1,H3K4me3 and H3K9me3 on TET1 promoter,respectively;besides,mRNA and protein level of TET1 was measured by qPCR and western blot,respectively;and MeDIP-qPCR was applied for detecting methylated level of TET1 promoter;while chromatin accessibility of TET1 promoter was evaluated by chromatin accessibility assay.ResultsPART1: On average,1,162,336 and 106,907 total examined CCGG sites were identified as 5mC and 5hmC modification in normal tissue,respectively,while 1,105,904 5mC and 75,236 5hmC modification on informative sites were observed in the CRC specimens.726 DMRs and 689 DhMRs between CRC group and normal tissue group were detected by HMST-seq.87.47% DMRs were hypermethylated,while 79.97% DhMRs were hypo-hydroxymethylated in CRC group,respect to normal tissue.83.88% of DMRs and 77.51% of DhMRs distributed in intergenic and gene body,while 13.22% of DMRs and 20.17% of DhMRs distributed in proximal regions close to TSSs and TSSs regions.Besides,53 DMR genes and 56 DhMR genes were selected as genes associated with CRC by bioinformatics analysis.Among these genes,ALOX15,GHRHR,TKTL1 and TFPI2 contained both DMR and DhMR in the same genomic position,which distributed in promoter or TSS regions.PART 2: Compared with control and empty vector groups,in H3R117 A LoVo cells: decreasing of both methylation and hydromethylation was observed on TFPI2 promoter,besides,more enrichment of TET1 and H3K4me3 but less enrichment of H3K9me3 were detected on TFPI2 promoter,while no difference was found in PAR level on this DNA fragment;on the other hand,expression of TET1 mRNA and protein level was boosted,and TET1 promoter was detected lower methylation and stronger chromatin accessibility,in addition,more enrichment of PAR and H3K4me3 but less PARP1 were detected on TET1 promoter,while no difference was found in H3K9me3 level.Conclusion1.Our study provided experimental evidence of genes with aberrant epigenetic modification in CRC and novel data for methylated and hydroxymethylated genomics of CRC.2.Mono-ADP-ribosylation of H3R117 regulated methylation of TFPI2 via TET1,promoting progress of CRC.Our study proved experimental evidence for mono-ADP-ribosylation of histone as a novel target for CRC therapy from epigenetic perspective. |
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